Folic acid intermediates



Patented Dec. 18, 1951 FOLIO ACID INTERMEDIATES David I. Weisblat,Barney J. Magerlein, and Arthur R. Hanze, Kalamazoo, Mich, assignors toThe Upjohn Gompany, Kalamazoo, Mich, a

corporation of Michigan No Drawing. Applicationllecember 3, 1948,

Serial No. 63,454

This invention relates to a method for the preparation ofN-((2-amino-4-hydroxy-6-pter idyDmethyl N (arylsulfonyl) p aminobenzoatecompounds and to certain intermediates useful in the method.

The N-((2 amino 4 hydroxy 6 pteridyl) methyl) N (arylsulfonyl) paminobenzoate compounds which can be prepared, by the method of theinvention have the generic formula COOR' wherein R is from the groupconsisting of hydro- 40 Claims. (Oi. 260-2515) gen and the alkylradicals and n is from the group consisting of zero and the positiveintegers l to '7, inclusive, and thus include N-substitution products ofp-aminoben-zoic acid, of p-aminobenzoyl glutamic acid, of thep-aminobenzoylglutamyl-glutamic acids having up to seven glutamic acid'residues in the molecule, and of their esters. In the naming ofcompounds having the generic formula given, which are described andclaimed in copending application Serial No. 41,882, filed July 31, 1948,nowPatent No. 2,558,711, and of other. starting compounds andintermediate compounds mentioned herein wherein both a glutamic acidresidue and a p-aminobenzoic acid residue are included in the molecule,the nitro gen atom of the glutamic acid residue is, for con- COORcompoundsused in or prepared by the method of the invention are thosewherein n of the generic formula given, and in those given subsequently,represents zero or the integer 1, i. e. those containing either noglutamic acid or ester residue or only one glutamic acid or esterresidue, and the method and intermediate compounds will be describedwith particular reference thereto.

Compounds having the generic formula given above and compounds useful asintermediates in the preparation thereof, are of particular value due tothe relationship of the former with, and to the ease with which they canbe converted to,

compounds similar to or identical with certain naturally occurringcompounds of the folic acid group. Thus, diethylN'-(N-((2-amino-4,-h-ydroxy 6 pteridyhmethyl) N (p toluenesulfonyl) paminobenzoyl) 1 glutamate can be converted readily by hydrolysis of thetwo ethyl ester groups with alkali to thecorresponding dicarboxycompound and the latter, by splitting the p-toluenesulfonyl radical fromthe molecule with hydrogen bromide and a bromine acceptor in analiphatic acid medium, according to the method described and claimed incopending application, Serial No. 41,883, filed July 31, 1948, nowPatent No. 2,562,222, can be converted readily to a pteroyl-glutamicacid which appears to be identical with the L. casei factor or vitaminBo from liver. The order of the hydrolysis step and splitting of thearylsulfonyl radical can,

be reversed, if desired.

Compounds having the generic formulae given in which the glutamic acidresidues have the l(+) configuration are of particular interest becauseof the existence of glutamic acid residues having the same configurationin folic acid compounds isolated from natural sources. The meth- 0d ofthe invention and the intermediate compounds to be described are,however, not limited as to the configuration of the glutamic acidresidues involved.

(Followed by hydrolysis when free acid is desired) N(3,3-dlalkoxy-2-hydroxypropyl) -N- (arylsulionyl)-p-amlnobenzoatecompound CEO-CHOH-CHr-NQCO (NHJJHC HaCHlGOhO R GOOR'oxidation Kri l-SO:

iv- (2-formyl-2-hydroxyethyl) -N (arylsu1fonyl) -p-aminbbenzate compoundCODE omoH-o O-CHz-NOC omncnomcmo ),.0R 011,0 on o O-GHz-NOC omnoncmomo o,on'

aryl-S 02 N- (3-l1ydroxy-2-ketopropy1) -N- (arylsulionyl)-p-aminobenzote compound aryl-S 0:

N- (3-methoxy-2-ketopropyl) -N-(arylsulfonyl) -p-aminobenzoate compoundoxidation I oxidation 1 1 C O O R OH CHO-C 0-CH-. NOC0(NH(JECH:CH1C o..oR' NH aryl- O; l

HQNXN NH:

III

N-(2-iormyl-2-ketoethyl)-N-(arylsulfonyl)-p-aminobenzoate 5 compound2,4,5'trlamlno-6-hydroxy- I I pyrimidine C O 0 R OH O o O R alkl-o-pon-o o-om-Noo 0 Nncnomomo o).0 R N CHYNOC O m mc 0) OR l aryk o, Il Taryl- O:

HaN- N M IV VIII 1! l -aminobenzoateN-((2-amino-4-hydroxy-fi-pteridyl)methyl) -N- (arylsullonyl) -p- N (3,3dlalkoxy 2 c m gom ony p aminobonzoate compound The process of theinvention, as illustrated in the accompanying reaction chart wherein theformulae of the compounds given are numbered to correspond with thefollowing description and wherein R and n have the values previouslygiven, comprises, among others, the step of reacting 'or condensing anN-(2-formyl-2-ketoethyl) -N-(arylsulfonyl) -p-amino-benzoate compound(III) with 2,4,5-triamino 6 hydroxypyo rimidine (VII) to form anN-((2-amino-4-hydroxy 6 pteridyl) methyl) -N-(ary1sulfony1)-paminobenzoate compound (VIII).

The N-(2-formyl-2-ketoethyl) N (arylsulfonyD-p-aminobenzoate compound(III) can be prepared in any one of several ways contemplated by theinvention. One such way comprises hydrolyzing the acetal group of anN-(3,3-dialkoxy-2- ketopropyl) -N-(arylsulfony1) -paminobenzoatecompound (IV) with an aqueous mineral acid. An N-(2-formyl-2-ketoethyl)-N- (arylsulfonyD- p-aminobenzoate compound (III) can also be preparedby oxidizing an N-(3-hydroxy-2-ket0- propyl) -N-(ary1sulfonyl)-p-aminobenzoate compound (V), e. g. with chromic or cupric acetate, inan aqueous alkanol. A further method for preparing anN-(2-formyl-2-ketoethyl) -N-(arylsulfonyD-p-aminobenzoate compound (III)consists in oxidizing an N-(3-methoxy-2-ketopropyl) N(arylsulfonyl)-p-aminobenzoate compound (VI), e. g. with chromicacetatein an aqueous medium. Still a further method for preparing an N-(Z-IQrmyI-Z-ketoethyl) -N- (arylsulfonyl) -p-aminobenzoate compound(III) con- 65 sists in oxidizing an N-(z-formyl-z-hydroxyethyl) -N-(arylsulfonyl) -p-aminobenzoate compound (II), e. g. with chromic orcupric acetate in an alcoholic or aqueous alcoholic medium.

An N-(3,3-dialkoxy-2-ketopropyl) -N-(arylsulfonyl)-p-aminobenzoatecompound (IV) can be prepared, as described and claimed in aconcurrently filed copending application, Serial No. 63,453, by heatinga t2,? -oxidopropana1 dialkyl acetal (IX) having the formula2,3-gxidoprqpanal dialkyl acetal with anN-(arylsulfonyl)-p-aminobenzoate alkyl ester (X) having the formulaN-(arylsulfonyl)-p-aminobenzoate alkyl ester followed by hydrolysis ofthe product with dilute alkali if the free acid is desired, to form an N-(3,3-dialkoxy-2-hydroxypropyl) N (arylsulfonyl)-p-aminobenzoatecompound (I) and subsequently oxidizing the latter hydroxy acetal, e. g.with chromic oxide, to form the keto acetal (IV).

An N- (2-formyl-2-hydroxyethyl) -N- (arylsulfonyl)-p-aminobenzoatecompound (II) can, as is herein first disclosed, be prepared by treatingan N (3,3-dialkoxy-2-hydroxypropyl) -N-(a.ry1- sulfonyl)-p-aminobenzoate compound (I) with an aqueous mineral acid, preferablyat an elevated temperature.

An N (3-hydroxy-2-ketopropyl) N arylsulfonyl) -p-aminobenzoate compound(V) can be prepared by first reacting an N-(arylsulfonyl)-paminobenzoate compound (XI) having the formula 000R H-N ooumoncmomo0),.01v

aryl-El 02 N-(arylsulfonyl)-p-aminobenzoate compound with anepihalohydrin (XII) Epihalohydrin according to the method described andclaimed in copending application Serial No. 41,884, filed propyl)1N-(.arylsulf0ny1) -'p-aminobenzoate compound (XIII) having the formula0 0 on hal-ClEh-CHOH-CHz-NQC o Nu-enongomo o n on aryl- O2 XIII jN-,(3-halo-2-hydroxypropyl) JN- (arylsulfonyl -p-an1inobenzoate compound Thelatter compound can then, as described and claimed in copendingapplication Serial No. 41,889, filed July 31, 1948, be oxidized withchromic oxide in glacial acetic acid to form an N (3-halo-2-ketopropyl)N (ary1sulfonyl)-paminobenzoate compound (XIV) having the formula XIV 0(@EHCHgO-Mg 'l Epoxypi'opyl aliphatic ester according to the methoddescribed and claimed in copending application Serial No. 41,890, filedJuly 31, 1948, to form an N(3-acyloxy-2-hydroxypropyl) -N-(arylsulfonyl)-p-aminobenzoate compound (XVI) having the formulaN-(3-acyIoxy-2-l1ydroxyp ropyl :N-(arylsulfonfl p-u minobenzoatecompound The latter compound can then, as described and claimed incopending application Serial No.' 41,889, previously referred to, beoxidized with ii chromic oxide to form an N-(3-acyloxy-2-ketopropyl) -N-(arylsulfonyl) -p-aminobenzoate com I pound (XVII) having the formulaN-(3-ucyloxy-2-ketopropyl -N- (a rylsul fonyl p-un1lnobenzoate compoundwhich, upon ester interchange with an alkanol, is converted to anN-(3-hydroxy-2-ketopropyl)- N-(arylsulfonyl) p aminobenzoate compound(V).

An N-(3-methoxy-2-ketopropyl) N (arylsulfonyl)-p-aminobenzoate compound(VI) can be prepared conveniently by treating an N-(3- halo 2hydroxypropyl) N (arylsulfonyl) psaminobenzoatej compound (XIII) with anaqueous alkali under carefully controlled condi tions, as described andclaimed in copending application Serial N 0. 41,884, previously referredto, to form an N-(2,3-epoxypropyl) N (arylsulfonyD-p-aminobenzoatecompound (XVIII) having the formula 0 000R o Hr HCHz-N oomnon'omouzc 0,.o11'

- aryl-S'O2 XVIII N- 2,3-epoxypropyl) -N- ('arylsulfonyl)-p-amiuobenzoate compound which can be reacted readily with sodiummethylate, according to the method described and claimed in the sameapplication Serial No. 41,884, to form anN-(3-methoxy-2-hydroxypropyl)-N- (arylsulfonyl) p aminobenzoate compound(XIX) having the formula coon I on, o o rn-ouo H-OHz-NQGO (NHcHomomoo,.o a

eryl-S 0g XIX N 3-methoxy-Z-hydroxypropyl) -N- (arylsulfonyl) -p-aminobenzoate compound The latter methoxy alcohol can be oxidized withchromic oxide by the method described and claimed in copendingapplication Serial No.v

41,889, previously referred to, to form an N-(3- methcxy 2ketopropyl)-N-(arylsulfonyl)-paminobenzoate compound. (VI) a TheN-(aryl'sulfonyl)-p-aminobenzoate com pounds (XI), including theN-(arylsulfonyl-paminobenzoate alkyl esters (X) are preparedconveniently by a method described and claimed in copending applicationSerial No. 41,888,-filed- July 31, 1948, which consists in reacting anarylsulfonyl halide with a p-aminobenzoate com-- pound (XX) havin theformula c 0 o R :NOG o Nndncmomc 0 ,.012

p-Aminobenzoate compound Of the starting materials used in the processof the present invention and intermediate compounds mentioned as usefulin the preparation thereof, certain of the N-(arylsulfonyl)-p-aminobenzoate compounds (XI) including certain of theN-(arylsulfonyl) p aminobenzoate alkyl.

esters (X), are described and claimed in copending application SerialNo. 41,888, filed July 31,.

1948; the N-(S acyloxy 2 hydroxypropyl) -N- (arylsulfonyl) paminobenzoate compounds (XVI), the N-(3-halo 2 hydroXypropyD-N-(arylsulfonyl) p aminobenzoate compounds (XIII), the N-(2,3-epoxypropyl)N (arylsulfonyl) -p-aminobenzoate compounds (XVIII), and theN-(S-methoxy 2 hydroxypropyD-N- (arylsulfonyl) p aminobenzoate compounds(XIX) are described and claimed in copending application Serial No.41,884, filed July 31, 1948;

the N-(3-acyloxy 2 ketopropyl)-N-(arylsul-' fonyl) -p-aminobenzoatecompounds (XVII), the

N-(S-halo 2 ke'topropyl)-N-(arylsulfonyl)-paminobenzoate compounds(XIV), the N-(3- methoxy 2 ketopropyl) N (arylsulfonyl) p-aminobenboatecompounds (VI) and the N-(3- hydroxy 2 ketopropyl), N (arylsulfonyD-p-aminobenzoate compounds (V) are described and claimed in copendingapplication Serial No 41,889, filed Jul 31, 1948; and the N-(3,3-dialkoxy 2 hydroxypropyl) -N-(arylsulfonyl)- p-aminobenzoate compounds.(I) and the N-(3,3-'

dialkoxy 2 ketopropyl) N (arylsulfonyl)- p-aminobenzoate compounds (IV)are described and claimed in a concurrently filed, copending applicationSerial No. 63,453.

The N-(2-formyl 2 hydroxyethyl) N-(arylsulfonyD-p-aminobenzoatecompounds (II) and the N-(2-formyl-2-ketoethyl) -N-(arylsulfonyl)p-aminobenzoate compounds (III) are new compounds first disclosed in thepresent application. They can be represented by the following genericformula (XXI) COOR aryl- O:

XXI

wherein Y is from the group consisting of radicals having the formulaeCHOH- and --CO- and n and R have the values previously given.

Esters which can be used as starting compounds in the process, with theproduction of the corresponding ester intermediates, include the methyl,ethyl, n-propyl, iso-propyl, butyl, amyl. hexyl, nonyl and other alkylesters. As a matter of convenience, alkyl esters containing less thanabout 8 carbon atoms in the alkyl ester radicals are preferably used inthe process, although insofar' as is known any alkyl ester can be used.

Although starting compounds containing substantially any aryls'ulfonylradical can be used in the process of the invention and in thepreparation of the corresponding arylsulfonyl intermediates and finalproducts, the preferred starting materials and intermediate products arethose containing the p-toluenesulfonyl radical due to the readyavailability of the p-toluenesulfonyl halides and to the generallycrystalline nature of the p-toluenesulfonyl derivatives of compoundswith which the present invention is concerned. Furthermore, it appearsthat the p-toluenesulfonyl radical can frequently be removed from thecompounds involved herein, by splitting with hydrogenbromide asmentioned previously, somewhat more readily and with the formation of asmaller proportion of undesired by-products than is the case with someof the other arylsulfonyl radicals. The invention is, however, notlimited to compounds containing the p-toluenesulfonyl radical andcompounds containing other arylsulfonyl radicals, such as theo-toluenesulfonyl, naphthalenesulfonyl, methylnaphthalenesulfonylradicals and others, can be used, if desired.

It should also be mentioned that compounds containing arylsulfonylradicals having nonhydrocarbon substituents can be used in the processand the corresponding intermediate and final products prepared, providedonly that the substituent is non-reactive under the reaction conditions.Such non-reactive substituents include chlorine, bromine, and themethoxy, phenoxy, nitro and similar radicals. It should be mentionedfurther that, although the present invention is concerned primarily withcompounds, wherein the sulfonyl derivative is an arylsulfonylderivative, the process can also be carried out and the correspondingintermediate and final Cal products prepared using starting materialscontaining alkylsulfonyl, aralkylsulfonyl or cyclo alkylsulfonylradicals, such as the methanesulfonyl, ethanesulfonyl,cyclohexylsulfonyl, and. phenylmethanesulfonyl radicals.

The conversion of an N-(3,3-dialkoxy-2-hydroxypropyl) -N- (arylsulfonyl)-p-aminobenzoate compound to an N-(2-formyl-2-hydroxyethyl)-N-(arylsulfonyl) -paminobenzoate compound is carried out convenientlyaccording to the method of the present invention by treating the hydroxyacetal with an aqueous mineral acid, preferably at an elevatedtemperature. The process can, insofar as is known, be carried out usingan N- (3,3-dialkoxy 2 hydroxypropyl) -N-(arylsulfonyD-p-aminobenzoatecompound containing any dialkyl acetal radical. From practicalconsiderations, however, the preferred compounds are those wherein thealkyl radicals of the dialkyl acetal group each contains less than 8carbon atoms. The reaction proceeds rapidly at or below the refluxingtemperature of the mixture and is generally complete in from a fewminutes to one hour or more. Satisfactory results have been obtained byheating the mixture for about fifteen minutes at about 60 C. Aqueousalcoholic mineral acid can also be used, if desired, and the reactionhastened by the greater solubility of the organic compound therein thanin aqueous mineral acid. Following the heating, the water and excessacid, together with any alcohol present in the mixture, can be distilledin vacuo and a residue obtained consisting of theN-(2-formyl-2-hydroxyethyl) N (arylsul fonyD-p-aminobenzoate compound ina form sufiiciently pure for most uses. Such of these hydroxy aldehydesas are esters can be hydrolyzed to the corresponding acids by carefulsaponification and acidification.

The oxidation of an N-(2-formyl-2-hydroxyethyl) -N- (arylsulfonyl)-p-aminobenzoate com pound to form an N-(2-formyl-2-ketoethyl)-N-(arylsulfonyl)-p-aminobenzoate compound can be carried out by heatingthe hydroxy aldehyde in an alcoholic or aqueous alcoholic medium withcupric acetate or chromic acetate. The oxidation is generallysubstantially complete in about one-half hour or less when the mixtureis heated at from about 50 to about C. In the case of oxidation usingcupric acetate, a precipitate of copper oxide is formed and this can beremoved readily by filtration. The alcohol or aqueous alcohol can thenbe distilled in vacuo and the N- (2-formyl-2-ketoethy1) -N-(arylsulfonyl)-p-aminobenzoate compound obtained as a residue which isgenerally in the form of a viscous, yellowish sirup.

When using chromic acetate as the oxidizing agent, the keto aldehyde canbe recovered from the mixture by evaporating the solvent and washing theresidue with water until the green color form an N-(2-formyl-2-ketoethy1) -N-(ary1su1- The free acids can be -fonyl)-p-aminobenzoate compound is carried out aldehyde compounds.

in an alcoholic or aqueous alcoholic medium using cupric acetate orchromic acetate in a manner substantially the same as that describedjust previously for the oxidation of the hydroxy The mixture isgenerally heated for from 5 to 30 minutes at from about 40 to about 80C. and the keto aldehyde then recovered from the mixture as previouslydescribed.

Oxidation of an N-i3-methoxy-2'-ketopropyl) N-(arylsulfonyl)-p-aminobenzoate compound to form an N-(2-formyl-2-ketoethyl) -N-(arylsulfonyl) -p-aminobenzoate compound is carried out using chromicacetate in a manner entirely analogous to that described for theoxidation of the hydroxy aldehydes and the hydroxy ketones in thepreceding paragraphs except that it is generally necessary to reflux theaqueous alcoholic reaction mixture for from one to several hours toinsure substantial completion of the oxidation reaction. The ketoaldehydes can be recovered from the reaction mixture using the methodspreviously described.

An N-(3,3-dialkoxy- 2 -ketopropyl)-N-(aryl- 'fcr from about 5 to about30 minutes'at from about 30 to about 80 C. The process can, insofar asis known, be carried out using an N-(3,3 dialkoxy 2 ketopropyD- N-(arylsulfonyl)-p aminobenzoate compound containing any dialkyl acetalradical. From practical considerations, however, the preferred compoundsare those wherein the alkyl radicals of the dialkyl acetal group eachcontains less than 8 carbon atoms. The keto aldehyde can be recoveredfrom the reaction mixture by evaporating the water and alcohol andwashing the oily residue free of acid with water.

Reaction of an N-(2-formyl-2-ketoethyl)-N (arylsulfonyl)-p-aminobenzoate compound with 2,4,5-triamino-6-hydroxypyrimidine toform an N((2-amino -4- hydroxy -6- pteridyll'methyD- N (arylsulfonyl).-p aminobenzoate compound can be carried out readily by mixing thesubstances, preferably together with anhydrous sodium acetate, inglacial acetic acid solution and allowing the mixture to stand for fromone to several hours at room temperature. The 2,4,5-triamino6-hydrcxypyrimidine is generally used in the form of itsdihydrochloride for greater ease in handling. The mixture is preferablystirred under an atmosphere of nitrogen or other inert gas. The reactionproceeds satisfactorily with the formation of the desired products insubstantial yield. It should be mentioned that, although the preferredconditions have been given, the reaction can be carried out underconditions differing substantially from these recited.

Following the reaction in acetic acid, the product can be recovered bydistilling the aceticacid in vacuo and washing the residue with water toremove traces of acetic acid and inorganic salts. TheN-((2-amino4-hydroxy -6- pteridyDmethyl)- N -(arylsulfonyl)- p-aminobenzoate compounds are thus obtained as highly colored residueswhich can, by the hydrolysis and cleavage procedure previously referredto, be converted into products containing a substantial proportion of afolic acid.

Among the N- (2-formyl -2- hydroxyethyl) -N- (arylsulfonyl)-p-amlnobenzoate compounds and N-(2-formyl -2-ketoethyl)-N-(arylsulionyl)-paminobenzoate compounds which can beprepared by the method herein disclosed are included N-(2 iormyl 2hydroxyethyl) -N-(ptoluenesulfonyl)-p-aminobenzoic acid, ethyl N-(2-f0rmyl -2- hydroxyethyl)- N -(p-toluenesulfonyl) p-aminobenzoate,n-propyl N-(2-formyl- 2 hydroxyethyl) N (alphanaphthylsulfonyl)p-aminobenzoate, butyl N-(Z-formyi-Z-hydroxyethyl)N-(p-chlorobenzenesulionyl)- p -aminobenzoate, amyl N-(Z-formyl -2-hydroxyethyh- N -(benzenesulfonyl) p aminobenzoate, N-(2- formyl- 2-hydroxyethyl) -N(p-nitrobenzenesulfonyl) -p-aminobenzoic acid, N'- (N-(2-formyl-2- hydroxyethyl) N -(p-toluenesulionyl) p -ami.- nobenzoyD-glutamic acid, diethyl N'-(N -(2 formyl 2 hydroxyethyD- N-(o-toluenesulfonyl)- p -aminobenzoyl)glutamate, di-n-butylN(N-(2-formyl- 2 -11 droxyethyl) N (p-ohlo robenzenesulfonyl) paminobenzoyl) l glutamate, N-(N-(2-I"ormyl 2 hydroxyethyl) -N-(naphthalenesulfonyhv aminobenzoyl) -g'lutamyl-glutamic acid,N(N(2-iormyl 2 hydroxyethyl) N p nitrobenzenesulfonyl) p aminobenzoyD-glutamyl glutamyl glutamic acid, N- (2-formyl 2 ketoethyl)-N-(p-toluenesulfonyl) -p-a'minobenzoic acid, ethyl N-(Z-formyI 2ketoethyD- N -(o-toluenesulfonyl)-paminobenzoate, n-propyl N-(2-formy1 2ketoethyl) N -(alpha naphthylsulfonyl) -p-aminobenzoate, butyl N-(2-formy1-2-ketoethyl) -N- (pchlorobenzenesulfonyl) -p aminobenzoate,amyl N-(Z-formyl 2 ketoethyD- N (benzenesulfonyl) -p-aminobenzoate,N-(2-formyl 4 2 ketoethyl) N -(p-nitrobenzenesulionyl)- p -aminobenzoicacid, N-(N-(2-formyl 2 ketoethyl)- N-(p toluenesulfonyD- p-aminobenzoyl) -glutamic acid, diethyl N'-(N-(2formyl 2 ketcethyl) -N-(o-toluenesulfonyl) -paminobenzoyl) glutamate, di-n-butylN-(N-)2-formyl-2-ketoethy1)- N -(p-chlorobenzenesulfonyl)-p-aminobenzoyl) -l-glutamate, N (N-(2-formyl-2-ketoethyl)- N-(naphthalenesu-lfonyl)- p aminobenzoyl) -glutamyl-glutamic acid, andN-(N(2- formyl 2 ketoethyD- N -(p-nitrobenzenesulfonyl) p -aminobenzoylglutamyl glutamylglutamic acid.

Certain advantages of the invention are apparent from the followingexamples which are given by way of illustration only and are not to beconstrued as limiting.

Example 1.--Ethyl N 3,3-dz'ethoary-2-hydroxy prom/l) N-(p-toluenesul;fonyl) p -amznobenzoate A mixture of one gram of thediethyl acetal of 2,3-oxidopropanal, 1.5 grams of ethylN-(ptoluenesulfonyl) -p-aminobenzoate and two drops of pyridine washeated for twelve minutes at I30-135 C. The clear melt which was formedwas cooled and seeded with crystals of previously prepared ethylN-(3L3-dietlroxy-2-hydroxypropy1)- N -(p-toluenesulfonyl)'- p-aminobenzoate.

The partially crystalline mass was triturated with a mixture of sixmilliliters of petroleum naphtha and three milliliters of isopropanoland the mixture filtered. There was thus obtained 1.71 grams of ethylN-(3,3-diethoxy-2-hydroxpyropyl) -N- (p-toluenesulfonyl)-p-aminobenzoate melting at 89-94 C. Repeated recrystallization of theproduct from isopropanol-petroleum naphtha raised the melting point to91-94 C. Hydrolysis '7 of the ester with dilute alkali yields N-(3,3-di-1 l ethoxy 2 hydroxypropyl) N -(p toluenesulfonyl) -paminobenzoic acid.

Example Z.--Ethyl N-(3,3-diethoaiy-2-ketopmpyl) -N- (p-toluene-sulfonyl)-p-aminobenzoate Approximately 3.2 grams of ethyl N-(ptoluenesulfonyD- p-aminobenzoate was reacted with the diethyl acetal of 2,3-oxidopropanalby the method described in Example 1. The crude reaction mixture priorto seeding was dissolved in milliliters of glacial acetic acid and thesolution mixed with a solution of one gram of 'chromic anhydride in 20milliliters of glacial acetic acid. The mixture was allowed to stand atroom temperature for about three hours and the acetic acid distilled invacuo. The residue was triturated with ether and the mixture filtered.The ethereal filtrate was washed with water, dried and the ethervolatilized. The residue consisted of ethyl N-(3,3-diethoxy-2-ketopropyl) N -(p toluenesulfonyl) p -aminobenzoate in the form of analmost colorless oil.

N-(3,3-diethoxy-2-ketopropyl) N -(p-toluenesulfonyl) -p-aminobenzoicacid is prepared by hydrolyzing its ethyl ester or by usingN-(3,3-diethoxy 2 hydroxypropyl) N -(p toluenesulfonyl)-p-aminobenzoicacid instead of its ethyl ester in the procedure just described.

Example 3.Ethyl N-(Z-formyl-Z-lcetoethyl)-- N- (p-toluenesulfonyl)-p-aminobenzoate About 600 milligrams of ethyl N-(3,3-diethoxy2-ketopropyl) -N-(p-toluenesulfonyl) p aminobenzoate is dissolved inabout 6 milliliters of methanol and the mixture diluted with 2milliliters of water containing 6 drops of concentrated aqueoushydrochloric acid. The solution is heated at about 60 C. for aboutminutes and the methanol and water distilled in vacuo. There is thusobtained a residue containing ethyl N-( 2 -formyl- 2 -ketoethyl) N -(ptoluenesulfonyl) -p-aminobenzoate.

N-(2-formyl 2 ketoethyl) -N-(p-toluenesulfonyl) p-aminobenzoic acid isprepared by carefully hydrolyzing its ethyl ester with dilute alkali orby using N -(3,3-diethoxy-2-ketopropyl) -N(ptoluenesulfonyl)-p-aminobenzoic acid instead of its ester in the aboveprocedure.

Example 4.Ethyl N-(Z-formyl 2 hydroxyethyl) N -(p toluenesulfonyl) p-aminobenzoate Six hundred milligrams of ethyl N-(3,3-diethoxy 2hydroxypropyD-N-(p toluenesulfonyl) -p-aminobenzoate was dissolvedin 6milliliters of methanol and diluted with 2 milliliters of watercontaining 6 drops of concentrated hydrochloric acid. The solution washeated at 60 C. for ten minutes and the methanol distilled in vacuo. Theoily residue was dissolved in ether, washed free of acid with water,dried and the ether distilled in vacuo. There was thus obtained 600milligrams of a light-colored oil containing a substantial proportion ofethyl N-(2-iormyl-2- hydroxyethyl) -N- (p-toluenesulfonyl) -paminobenzoate.

N -(2-formyl-2-hydroxyethyl) -N- (p toluenes'ulfonyl) -p-aminobenzoicacid is prepared by hydrolyzing its ethyl ester with dilute alkali or byusing N 3,3-diethoxy-2-hydroxypropyl) N- ptoluenesulfonyl)-p-aminobenzoic acid instead of its ester in the above procedure.

12 Example 5.Ethyl N-(2-formyl-2-ketoethyl) -N- (p-toluenesuljonyl)-p-aminobenzoate The oily product from Example 4 was dissolved in 20milliliters of methanol and a solution of 300 milligrams of cupricacetate in 2 milliliters of water was added. The mixture was heated atabout 60 C. for ten minutes. The copper oxide which precipitated wasseparated by filtering and the water and alcohol were distilled from thefiltrate in vacuo. The residual oil, after dryi'ng'in vacuo, weighed 600milligrams and contained a substantial proportion of ethylN-(2-formyl-2- ketoethyl) N- (p-toluenesulfonyl) p -aminobenzoate in theform of a viscous, yellow sirup.

N-(2-formyl 2 ketoethyl) -N-(p'- toluenesulfonyl) -p-aminobenzoic acidis prepared in analogous manner using N-(2-'formyl-'2-hydroxyethyl)-N-(p toluenesulfonyl) -p aminobenzoic acid instead of its ester.

Example 6.-Ethyl N (3 chloro 2 hydromypropyl) N (p toluenesulfonyl) paminobenzoate A mixture of five grams of ethylN-(p-toluenesulfonyl)-p-aminobenzoate and 3.4 milliliters ofepichlorohydrin was heated at C. and two drops of pyridine added. Avigorous action ensued and after five minutes the mixture was cooled,dissolved in 50 milliliters of ethanol and treated three times withdecolorizing carbon. The ethyl N- (3-chloro-2-hydroxypropyl)-N-(p-toluenesulfonyl)-p-aminobenzoate which remained uponvolatilization of the ethanol and excess epichlorohydrin in vacuo wasused in subsequent reactions without further purification.

Example 7.Ethyl N- (3-chloro-2-ketopropyl) N- (p-toluenesulfonyl)-p-aminobenzoate The crude oily ethyl N-(3-chloro-2-hydroxypropyl)-N(p-toluenesulfonyl) p-aminobenzoate prepared from 30 grams of ethylN(-p-toluenesulfonyl) -p-aminobenzoate and an excess of epichlorohydrinwas dissolved in milliliters of acetic acid and a mixture of 12 grams ofsodium dichromate, 10 milliliters of sulfuric acid, 45 milliliters ofwater and 60 milliliters of acetic acid was added over a period of threehours while maintaining the mixture at 5 C. After stirring for anadditional three hours, the oxidation mixture was diluted with water andextracted with ether. The ethereal extract was washed with sodiumbicarbonate and the ether distilled. The residue of ethyl N-(3-chloro-2-ketoprppyl) -N-(ptoluenesulfonyl) p aminobenzoatecrystallized from diluteethanol on prolonged standing. The crystallizedproduct weighed 5.5 grams and after two crystallizations from diluteethanol, melted at 106 to 113 C.

Example 8.-Ethyl N-(3 hydroxy 2 ketopropyl) N (p toluenesuljonyl) paminobenzoate One gram of ethyl N-(3-chloro-2-ketopropyl)-N-(p-toluenesulfonyl) -p-aminobenzoate was dissolved in 20 millilitersof acetone and the solution diluted with 10 milliliters of water.One-half gram of barium carbonate was then added and the mixture stirredovernight at room temperature and then refluxed for two hours. Themixture was then filtered, partially concentrated, refiltered and thenconcentrated. The thick liquid was dissolved in ether, washed with waterand again concentrated to a viscous liquid. The ethyl N-(3-hydroxy-2-ketopropyl) N (p'- toluenesultate instead of cupricacetate.

13 fony1)-p-aminobenzoate thus obtained weighed 0.27 gram.

The ester can be hydrolyzed by careful saponi- -fication to form thefree N-(3-hydroXy-2-ketopropyl) N-(ptoluenesulfonyl) p aminobenzoicacid.

Example 9.-Ethyl N (Z-jormyl-Z-lcetoethyl) N (p-toluenesulfonyl)-p-aminobenzoate A mixture consisting of 770 milligrams of ethylN-(3-hydroxy-2-ketopropyl) N (p toluenesulfonyl) -p-aminobenzoate, 284milligrams of cupric acetate, 20 milliliters of methanol and 2milliliters of water was heated at about 60 C. for minutes. Theprecipitated copper oxide was removed by i'iltration and the filtratewas distilled in vacuo to remove the methanol. The residue consisted ofan aqueous and an oily layer. The oily layer was separated and dissolvedin ethyl acetate and the copper salts Washed from the solution withwater. I

The ethyl acetate solution was then dried with sodium sulfate and thesolvent removed by distil- Example 10. EthylN-((Z-amino--hydrory-fipterz'clybmethyl) N (p toluenesulfonyl)-paminobenzoate Six hundred milligrams of crude ethyl N-(2-formyl-2-ketoethyl) -N -(p toluenesulionyl) paminobenzoate was dissolvedin milliliters of glacial acetic acid and the solution was added to amixture of 150 milligrams of 2,4,5-triamino-6- hydroxypyrimidinedihydrochloride and 115 milligrams of anhydrous sodium acetate. Themixture was stirred for ninety minutes at room temperature under anatmosphere of nitrogen and the acetic acid was then removed from themixture by distillation in vacuo. There was thus obtained a highlycolored residue containing inorganic salts and ethyl N ((2 amino 4hydroxy 6- pteridyl) methyl) N (p toluenesulfonyl) paminobenzoate.

In entirely analogous fashion 2,4,5-triamino-6- hydroxypyrimidine isreacted with N- (2-formyl-2 ketoethyl) -N- (p-toluenesulfonyl) paminoben- ,zoic acid to form N ((2 amino 4 hydroxy- 6 pteridyl) methyl)N (p toluenesulfonyl) p-aminobenzoic acid.

Example 11 .-Hyd.rolysis and cleavage of ethyl N- ((2 amino 4 hydroayd-pteridyl) methyl) N- ('p-toluenesulfonyl) -p-aminobeneoate The crudeethyl N-((2-amino-l-hydroxy-6- pteridyl) methyl) N (p toluenesulfonyl)paminobenzoate was dissolved in 3.5 milliliters of a 26 per cent byweight solution of hydrogen bromide in glacial acetic acid and 0.13 gramof phenol was added quickly. The mixture was stirred at room temperaturefor one hour and then poured into 30 milliliters of anhydrous ether.After mixing thoroughly and allowing to stand for a short time, themixture of ether and suspended solid material was filtered and theresidue washed twice with anhydrous ether and then dried in vacuo forseveral hours. The dry residue consisted of impure ethyl N-((2-amino-4.-hydroxy-6,

'pteridyl) methyl) -p-aminobenzoate. The product was hydrolyzed withdilute sodium hydroxide to convert the esterto the free acid. .Theproduct had an activity when bio-assayed with Streptococcus fecalis R.corresponding to a. purity of 32 per cent ofN-((2-amino-4-hydroxy-6-pteridyl)- methyl) -p-aminobenzoic acid.

Example 12.-Diethyl N-(N-(p toluenesulfonyl) -p-amz'nobenzoyl)-l-glutamate Thirty and nine-tenths. grams of N-(p-t0luenesulfonyl) paminobenzoyl chloride and 23.9 grams of diethyl l(+) glutamatehydrochloride were dissolved in 300 milliliters of, ethylene dichlorideand the solution cooled to between 0 and 10 C. The cold solution wasstirred vigorously and 22.3 grams of triethylamine in 72; milliliters ofethylene dichloride was added slowly over a period of about 20 minutes.The temperature of the mixture was held between 10 and 720 C. during theaddition of the triethylamine and the mixture then allowed to stand atroom temperature for one hour. The mixture was then washed successivelywith water, dilute hydrochloric acid, saturated aqueous sodiumbicarbonate and finally with water. The colorless solution thus obtainedwas dried with anhydrous sodium suliate and naphtha was added until thesolution became opalescent. The mix.- ture was then cooled to causecrystallization and filtered. The crystals, after drying, consisted of36 grams of diethyl N'-(N-(p-toluenesulfonynp-aminobenzoyl')-l-glutamatemelting at 124 to 126 C.

Example 13.Diethyl N '-(N- (3,3-diethoxy-2-hydroxypropyl)-N-(p-toluenesulfonyl) p aminobenzoyl) -l-glutamate One and six-tenthsgramsof the diethyl acetal of 2,3-oxidopropanal and five drops ofpyridine were added to 4.77 grams of fused diethyl N"-('N-(p-toluenesulfonyl) p aminobenzoyl) -l-glutamate at C. The mixture was:stirred for about 30 minutes at to C. The highly colored mass consistedchiefly of diethyl N-(N- (3,3 diethoxy-2-hydroxypropyl)-N.-(p-t0luenesulfonyl) -p-aminobenzoyl) -l-g1utamate. It had an indexof refraction without further purification of N =1.527.N.(N-(3,3-diethoxy-2-hY- droxypropyl) -N (p toluenesulionyl) -p-amino.-benzoyl) -l-g1utamic acid is formed. by hydrolyz ing the ester withdilute alkali.

Example 14.--Diethyl N '-(N (3,3 diethozcy 2- ketopropyD-N-(ptolaenesulfonyl). p-ami'no benezoyl) -l-glutamate Five grams of diethylN-(N-(p-toluenesulfonyl) -p-aminobenzoyl) -1-glutamate and 1.9 grams ofthe diethyl acetal of 2,3-oxidopropanal were reacted substantially as bythe method of Example 13. The crude diethyl N'-(N-(3,3-diethoxy-2-hydroxypropyl) -N- (p-toluenesulfonyl) -p-aminobenzoyl) -1-glutamatethus obtained was dissolved in 35 milliliters of glacial acetic acid and1.7 grams of chromic anhydride was added. to the solution. The mixturewas stirred at room temperature for about two hours and then filtered.The filtrate was diluted with water and extracted with a mixture ofbenzene and ethyl acetate. The organic layer was separated, washed freeof acid with water and dried over anhydrous sodium sulfate. The solventwas then distilled in vacuo and there was thus obtained 5.9 grams ofdiethyl N'-(N-(3,3-diethoxy-2-ketopropyl) N .(p-toluenesulfonyl) paminoben zoyD -1-glutamate as a of the ester with dilute alcoholicsodium hydroxyellow sirup. Hydrolysis ide forms N (N-(3,3-diethoxy-2-ketopropyl) I- (p -toluenesulfonyl)-p-aminobenzoyl) lglutamic acid.

Example 15.-Diethyl N '-(N -(2-f0rmyl 2 ketcethz l)-N-(p-toluenesuljonyl) p amlnobenzoyl) -l-glutamate A mixture consistingof 1.5 grams of diethyl -N-(N-(3,3-- diethoxy-2-ketopropyl) N-(ptoluvacuo. The residue consisted of 1.1 gram of diethyl N' (N-(2-formyl-2-ketoethyl) -N- (p-toluenesulfonyl) -p-aminbenzoyl) lglutamate in the form of a viscous mass.

N (N- (2 formyl-Z-ketoethyl) -N- (p-toluenesulfonyl) p aminobenzoyl)-l-glutamic acid is prepared by careful hydrolysis of its diethyl esterwith dilute alkali or by heating N(N-(3,3-diethoxy-Z-ketopropyl)-N-(p-toluenesulfonyl) paminobenzoyl) -l-glutamic acid with aqueousmineral acid as described above.

Example 16.Diethyl N-(N-(2- ormyl 2 hydroxyethyl) -N-(p toluenesulfonyl)-p-amlnobenzoyl) -l-glutamate The crude diethylN-(N-(3,3-diethoxy-2-hydroxypropyl) N- (p-toluenesulfonyl) paminobenzoyD-l-glutamate obtained in Example 13 was dissolved in amixture consisting of 25 milliliters of methanol, milliliters of waterand 1 milliliter of concentrated aqueous hydrochloric acid. The mixturewas refluxed for 25 minutes and the methanol distilled in vacuo. Theresidue was dissolved in ethyl acetate and the solution washed withwater and dried. Distillation of the ethyl acetate in vacuo gave 5.2grams of a residue containing a substantial proportion of diethyl N- (N(2 formyl-Z-hydroxyethyl) -N-(p-to1uene sulfonyl) -p-aminobenzoyl) lglutamate in the form of a light-colored, viscous oil. The oil gave ayellow color with aqueous alkali and a positive test with Fehlingssolution- N (N (2-formyl-2-hydroxyethyl) N (p-toluenesulfonyl)-p-aminobenzoyl) -l-glutamic acid is formed by careful saponification ofits diethyl ester or by heating N-(N-(3,3-diethoxy-2-hydroxypropyl)-N-(p-toluenesulfonyl) p-aminobenzoyl)-l-glutamic acid with aqueousmineral acid as described above.

Example 17.-Diethyl N'-(N-(2formyl-2-ketoethyD- N(p-toluenesulfonyl)-p-aminoben zoyl) -l-glutamate Five grams of diethylN-(N-(2-formyl-2-hydroxyethyl)-N-(p-toluenesulfonyl) p aminobenzoyl)-l-glutamate was dissolved in 50 milliliters of methanol and thesolution mixed with a solution of 1.8 grams of cupric acetate in amixture of 50 milliliters of methanol and 10 milliliters of water. Themixture was heated at 60 C. for twelve minutes, the copper oxide wasremoved by filtering and the filtrate was distilled in vacuo to removethe methanol. The residue was dissolved in ethyl acetate and thesolution was washed with water, dried and the ethyl acetate distilled invacuo. There was thus obtained 4.6 grams of a residue containing asubstantial proportion of diethyl N'-(N- (2-formyl-2-ketoethyl)-N-(p-toluenesulfonyl) -p-aminobenzoyl) -l-glutamate in the form of ayellow sirup. The product gave a yellow color with aqueous alkali and apositive test with Fehlings solution. Similar results are obtained usingchromic acetate instead of cuprlc acetate as the oxidizing agent.

N -(N-(2-formyl-2-ketoethyl) N (p-toluenesulfonyl) -p-aminobenzoyl)-l-glutamic is formed by using N (N- (2-formyl-2-hydroxyethyl) N-(ptoluenesulfonyl)-p-aminobenzoyl l glutamic acid instead of its diethylester in the oxidation procedure described above.

Example 18.2,3-epo:vypropyl acetate (glycidol acetate) A mixture of 98grams of anhydrous potassium acetate, 10 grams of potassium carbonateand 250 milliliters of dry epichlorohydrin was refluxed for 25 hours.The mixture was cooled, filtered, and the filtrate fractionallydistilled. A first fraction of unreacted epichlorohydrin was recoveredand a fraction consisting of 73 grams of 2,3-epoxypropyl acetate boilingat 82 to 84 C. under a pressure of 40 millimeters was collectedseparately, The compound had an index of refraction ND =1.4201.

Example 19.-Diethyl N (N-(3-acetoxy-2-hydrozcylpropyl) -N-(p-toluenesulfonyl) -p-aminobenzoyl) -glutamate A mixture of 59.3 gramsof diethyl N '-(N(ptoluenesulfonyl) p aminobenzoyl)-glutamate and 12.7grams of 2,3-epoxypropyl acetate was heated at 150 C. until a clear meltwas formed. Five drops of pyridine were then added and the mixtureheated at to C. for about onehalf hour. The sirupy reaction mixture wasthen dissolved in 100 milliliters of benzene and the solution washedwith water and dilute hydrochloric acid. The solution was dried andchromatographed on 1200 grams of alumina. The product was eluted with asolution of 5 per cent of ethanol in benzene. Evaporation of the alcoholand benzene gave a residue of 66 grams of diethyl N (N(3-acetoxy-2-hydroxypropyl) -N- (p toluenesulfonyl -p-aminobenzoyl)-glutamate in the form of an oil having an index of refraction ND=1.5396.

Example 20.-Dz'ethyl N-(N-(3-acetomy-J2-lcetopropyl) N (ptoluenesulfonyl)-p-aminobenzoyl) -glutamate A mixture was preparedconsisting of 8.86 grams of diethyl N (N-(3-acetoxy-2-hydroxypropyl) N-(p-toluenesulfonyl) -p-aminobenzoyl) -glutamate having an index ofrefraction N =1.5396, 50 milliliters of glacial acetic acid and 10milliliters of propionic acid. The mixture was cooled to 0 C. and a.solution of 1.65 grams of chromic anhydride in a mixture of 1.5milliliters of water and 30 milliliters of glacial acetic acid was addedslowly with stirring. The mixture was allowed to stand at about 5 C. fortwelve hours and the solvent then distilled under reduced pressure. Theresidue was treated with a mixture of water and ether and the layersseparated. The ether layer was washed twice with saturated sodiumchloride solution then with saturated sodium bicarbonate solution andagain with saturated sodium chloride solution. The washed etherealsolution was then dried with anhydrous magnesium sulfate and the etherdistilled. There was thus obtained 6.1 grams of yellowish oily diethyl N-(N -.(3 -acetoxyenesulfonic acid and s milliliters of absolute wethanol was refluxed for three hours and then distilled slowly untilmilliliters of distillate had been collected. An additional 40milliliters of absolute ethanol was added and the mixture again refluxedfor 2.25 hours. The mixture was then concentrated in vacuo and thesirupy residue taken up in ether and washed successively with 3 portionsof water and one portion of saturated sodium chloride solution. Theethereal solution was then dried over anhy .lrous magnesium sulfateThere was and the ether evaporated in vacuo. thus obtained 2 grams ofviscous diethyl N-(N- (3 hydroxy 2ketopropyl)-N-(p-toluenesulfonyD-p-aminobenzoyl)-1-glutamate having aspecific rotation at 25 C. in absolute ethanol of l8. The product can bepurified and obtained in solid form using Girards reagent P. Carefulhydrolysis of the ester with dilute alkali gives N-(N-(3-hydroxy-2-ketopropyl) N (p toluenesulfonyl) p-aminobenzoyl)-glutamic acid.

Example 22.'Diethyl N-(N-(2-jo1'myl-2-lcetoethyl) -N(p toluenesulfonyl)p aminobenzoyl) -l-glutamate A solution of 300 milligrams of soliddiethyl N (N-( 3-hydroxy-2-ketopropyl) -N -(p-to1uenesulfonyl)-p-aminobenzoyl) l glutamate in milliliters of absolute ethanolcontaining a catalytic amount of p-toluenesulfonic acid and sufficientchromic acetate to give the solution a permanent green color wasrefluxed for 3 hours. The solution was then distilled in vacuo to removemost of the ethanol and the organic portion of the residue was dissolvedin ethyl acetate. The ethyl acetate solution was washed with water anddried and the ethyl acetate distilled in vacuo. There was thus obtained290 milligrams of oily residue containing a substantial proportion ofdiethyl N-(N (2 formyl-Z ketoethyl) N -(p toluenesulfonyl) -paminobenzoyl) -1-glutamate. Similar results were obtained using crude,oily diethyl N-(N-(3-hydroxy-2-ketopropyl) N -(ptoluenesulfonyl)-p-aminobenzoyl -l glutamate which had not been purified using Girardsreagent.

N-(N-(2-formyl-2-ketoethyl) N -(p-toluenesulfonyl) p aminobenzoyl) lglutamic acid is formed by oxidation of N'-(N-(3hydroxy-2-ketopropyll-N-(p toluenesulfonyl) p aminobenzoyl) l-glutamic acid withchromic acetate by the procedure just given.

Example 23.Diethyl N'-(N-(2-formyl-2-ketoethyD-N-(p toluenesulfonybpaminobeneoyl) -Z-glutamate A solution of 350 milligrams of solid diethylN'- (N- (3-hydroxy-2-ketopropyl) -N-(p-toluenesulfonyl)-p-aminobenzoy1-l-glutamate and 142 milligrams ofcupric acetate in a mixture of 20 milliliters of methanol and 2milliliters of water was heated at 60 C. for 10 minutes. The solutionwas filtered to remove precipitated copper oxide and then distilled invacuo to remove methanol. The residue was dissolved in ethyl acetate andthe solution was washed well with water, dried with sodium sulfate anddistilled in vacuo to remove the ethyl acetate. There was thus obtained330 milligrams of an oily residue containing a substantial proportion ofdiethyl N-(N -(2-formyl-2-ketoethyl) N (p toluenesulfonyl)-p-aminobenzoyl) l -glutamate. Similar results were obtained usingcrude, oily diethyl N-(N -(3 hydroxy-Z-ketopropyl) N -(ptoluenesulfonyl) -p-aminobenzoyl) l glutamate which had not beenpurified using Girards reagent.

N'-(N -(3-hydroxy-2-ketopropyl) N -(p-toluenesulfonyl) -p-aminobenzoyl)l glutamic acid is oxidized with cupric acetate in similar manner toform N-(N-(2-formyl-2 ketoethyl) N -(p toluenesulfonyl) p aminobenzoyl)l l glutamic acid.

Example 24.-Diethyl N'-(N -(3 methomy 2 hydromyp'ropyb- N ."ptoluenesulfonyD- p aminobenzoyl) -l-ylutamate A mixture of 7.2 grams ofdiethyl N-(N-(ptoluenesulfonylp -ar ninobenzoyl) l glutamate and 1.45grams of 1,2-epoxy-2-methoxypropane was heated in an oil bath at C.until melted and five drops of pyridine then added. The mixture wasstirred at 145 to for thirty minutes and then dissolved in 50milliliters of benzene. The benzene solution was washed three times with20 milliliter portions of dilute sulfuric acid and then with water untilthe washings were neutral. The benzene solution was then dried withanhydrous sodium sulfate, treated twice with 0.5 gram portions ofdecolorizing charcoal and filtered. The benzene was then evaporated fromthe filtrate under reduced pressure and there were thus obtained 8.0grams of diethyl N- (N- 3-methoxy-2-hydroxypropyl) N -(ptoluenesulfonyl) p aminobenzoyl) l glutamate as an almost colorless,somewhat viscous liquid having an index of refraction N =l.538.

In similar fashion ethyl N-(p-toluenesulfonyD-p-aminobenzoate is reactedwith 1,2- epoxy-B-methoxypropane to form ethyl N-(3- methoxy 2-hydroxypropyl) N -(p-toluenesulfonyl) -p--aminobenzoate.

Example 25.-Diethyl N '-(N 3-methomy-2-lcetoproml)-N-(p-toluenesul,fonyl)-p aminobenzoyl) -lglutamate A solution of0.536 gram of chromic oxide in about 5 milliliters of water was addedslowly at 20 C. and with stirring to a solution of 2 grams of diethylN'-(N-(3-methoxy-2-hydroxypropyl) N -(p toluenesulfonyl) paminobenzoyl)-l-glutamate in 19 milliliters of glacial acetic acid. Themixture was allowed to stand for three and one-half hours and was thenpoured into water and extracted with benzene. The benzene layer waswashed thoroughly with water and the benzene vaporized in vacuo. Therewas thus obtained a residue consisting of 1.3 grams of diethylN'-(N-(3methoxy-2- ketopropyl) N -(p-t o1uenesulfonyl)- paminobenzoyl)-l-glutamate having an index of refraction N =L54i0 andmelting at 82 to 84 C.

In a similar manner ethyl N-(B-methoxy-Z- hydroxypropyl) N -(ptoluenesulfonyl) -p aminobenzoate is oxidized with chromic oxide to formethyl N-(3-methoxy-2-ketopropyl)-N- (p toluenesulfonyl) p-.aminobenzoate. N-("- methoxy-2-ketopropyl) N (p-toluenesulfony1)-p-aminobenzoic acid and N-(N-(3-methoxy-2- ketoprcpyll-N-(ptoluenesulfonyD-p-aminobenzoyD-l-glutamic acids are formed by carefulsaponification of their respective ethyl esters.

Example 26. Diethyl N-(N-(2-formyl-2-lcetoethyl) N (p toluenesalfonyl)-p-aminoben-- zoyl) l-glutamate (N (2 formyl 2-ketoethyl)N-(p-toluenesulfonyl) p-aminobenzoyl) l-glutamate which was used in asubsequent experiment without further purification.

In a similar manner, ethyl N-(3-methoxy-2- ketopropyl) N (ptoluenesulfonyl) p-aminobenzoate is oxidized by chromic acetate with theformation of ethyl N-(2-formyl-2-ketoethyl)N- (p-toluenesulfonyl)p-aminobenzoate.

N (2 formyl 2 ketoethyl) N-(p-toluenesulfonyl)-p-aminobenzoic acid andN-(N(2- formyl 2 ketoethyl) N-(p-toluenesulfonyl) paminobenzoyl)l-glutamic acid are formed by careful saponification of their ethylesters or by oxidizing N (3methoxy-Z-ketopropyl)N-(ptoluenesulfonyl)p-aminobenzoic acid or N'-(N- 2(3 methoxy 2 ketopropyl)N-(p-toluenesulfonyl) p-aminobenzoyl) l-glutamicacid, respectively, with ohromic acetate according to the method justdescribed.

Example 27. Diethyl N (N-((2-amino-4-hydroxy 6 pteridybmethyl)N-(p-toluenesulfonyl) p-otminobenzoyl) l-glutamate Three hundred thirtymilligrams of crude diethyl N (N (2 formyl 2 ketoethyl)N-(ptoluenesulfonyl) p-aminobenzoyl)-l-glutamate was dissolved in 11milliliters of glacial acetate acid and the solution mixed with 107milligrams of 2,4,5-triamino-6hydroxypyrimidine dihydrochloride and 82milligrams of sodium acetate. The mixture was stirred at roomtemperature under an atmosphere of nitrogen for about ninety minutes andthen concentrated in vacuo to remove most of the acetic acid. Washing ofthe residue with water gave a product consisting principally of diethylN'-(N-((2-amino-4-hydroxy 6 pteridyl)methyl) N (p-toluenesulfonyl)p-aminobenzoyl) l-glutamate.

2,4,5triamino6-hydroxypyrimidine is reacted with N (N (2 formyl 2ketoethyl) N-(ptoluenesulfonyl) p aminobenzoyl) l-glutamic acid in amanner similar to that just described to form N (N((2amino4-hydroxy-6-pteridyl) methyl) N (p toluenesulfonyl)p-aminobenzoyl)-l-glutamic acid.

Example 28.-Hydrolysis and cleavage of diethyl N (N ((2 amino 4hydromy-G-pteridyl) methyl) N-(p-toluenesulfonyl) p-amlnobenzoyl)l-glatamate mixed with 130 milligrams of phenol and 2.5

milliliters of a 26 per cent solution of hydrogen bromide in glacialacetic acid. The mixture was stirred for ninety minutes at roomtemperature and then diluted with anhydrous ether. Ethyl pteroylglutamate hydrobromide separated from the mixture as a brown,hygroscopic solid which was recovered by filtration. The brown solid wasdissolved in about two milliliters of methanol and ten per cent aqueoussodium hydroxide was added to give a strongly alkaline solution. Thealkaline mixture was allowed to stand for ninety minutes at 40 C. andthen acidified with mineral acid to pH 3.0. The solution was thendiluted with five milliliters of water, cooled in an ice bath for onehour and then centrifuged. The

solid residue was washed with three milliliters of water and three timeswith three milliliters of acetone and dried in vacuo over sulfuric acid.There was thus obtained 89 milligrams of pteroyl glutamic acid which,upon microbiological assay with Lactobacillus casei had an activity of48.8 per cent of that of the pure natural acid.

We claim:

1. A compound having the formula COOR aryl-SO2 wherein R is from thegroup consisting of hydrogen and the alkyl radicals, n is from the groupconsisting of zero and the positive integers 1 to 7, inclusive, and Y isfrom the group consisting of radicals having the formulae CHOH- and CO.

2. A compound as claimed in claim 1 wherein R is hydrogen.

3. A compound as claimed in claim 1 wherein R is an alkyl radical.

4. A compound as claimed in claim 1 wherein n is zero.

5. A compound as claimed in claim 1 wherein n is the positive integer 1.

6. A compound as claimed in claim 1 wherein the arylsulfonyl radical isthe p-toluenesulfonyl radical.

'7. A compound having the formula COOR l aryl-S O 2 wherein R is fromthe group consisting of hydrogen and the alkyl radicals and n is fromthe group consisting of zero and the positive integers 1 to '7,inclusive.

8. Ethyl N- (2-formyl-2-hydroxyethyl) N- (ptoluenesulfonyl)p-aminobenzoate.

9. Diethyl N (N- (2-formyl-2-hydroxyethyl) N- (p-toluenesulfonyl)p-aminobenzoyl) glutamate.

10. A compound having the formula OOOR wherein R is from the groupconsisting of hydrogen and the alkyl radicals and n is from the groupconsisting of zero and the positive integers 1 to 7, inclusive.

11. Ethyl N-(2-formyl-2-ketoethyl) N (D- toluenesulfonyl)p-aminobenzoate.

12. Diethyl N-(N-(2 formyl-Z-ketoethyl) N- (p toluenesulfonyl)paminobenzoyl) glutamate.

tea

$3. The method which includes: reacting a wherein R1 is from the groupconsisting ofhyketo aldehyde having the formula I, drogen and the alkylradicals and. n is from the COOK, group consisting of zero and thepositive integer .CHO;C;CH2 NC CO(NEHC,HZGHaCO) OR, a 1,, to form a ketoaldehyde haingiglhe formula arylws'm UB 00 CH N 00 NHJJHCH CHCO) ORwherein R" is from the group consistingof hy-' I i drogen and the alkylradicals and 71 isfrom the Q1 group consisting of zero and the positiveinteger and, subsequently, reacting the latter compound 1, with2,4,5-triamino-G-hydroxypyrimidine to with2,4,5-triamino-dehydroxypyrimidine toiform form a compound having theformula a compound having the formula OH 0H N (500111 N 000R N om-iiocomnoncmomo o ..on' N -om-ucoomnoncmcmo O),.0It L aryl-SOa, V aryl-AO:I a N j i 'N N wherein R" and n have the Values given. R andnhaving thevalues given.

14. The method of claim 13 wherein n is zero. 25. The method of claim 24wherein the hy- 15. The method of claim 13 wherein n is the dmoxyaldehyde is ethyl N-(Z-formyl-Z-hydrOXY- positive integer 1. ethyl) N (ptoluenesulfonyl) p amino- 16. The method of claim 13 wherein R ishybenzoate. drogen. 26. The method of claim 24 wherein the hy- 17. Themethod of claim 13 wherein R. is an droxy aldehyde is diethy-lN'--(N-(2'-formyI-2-.hy+ alkyl radical. droxyethyl) N (ptoluenesulfonyl) p- 18. The method of claim 13 wherein thearylaminobenzoyl) -glutamate. sulfonyl radical is the p-toluenesulfonylradical. 27. The. method of claim 24 wherein the hy- 19. The method ofclaim 13 wherein the keto droxy aldehyde is oxidized with. cupricacetate. aldehydeds ethyl y 28. The method of claim 24 wherein the hypv1-pat droxy aldehyde is-oxidized with chromic acetate. 20. The method ofclaim 13 wh rein the k o 29. The; method which includesz-hydrolyzing aaldehyde is diethyl N (N (2-formyl-2-ketohydrqxy acetal having theformula Goo'w (alky1-O-);CHOHOHCHN 10 o (NHHGHzCHaCOhO n ary1-S Oz I Hethyl) N (p toluenesulfonyl) p aminoben wherein R is from the groupconsisting of hyzoyl) -glutamate. drogen and the alkyl radicals and n isfrom the 21. The method which includes: hydro-lyzing i0 group consistingof zero and the positive integer a keto acetal having the formula l a gCOOR aik 1-0-)2cH-oo-cm-1 woomnoncmomo 0),.oR'

aryl-SOZ wherein R is from the group consisting of hy- 1, to form ahydroxy aldehyde having the fordrogen and the alkyl radicals and n isfrom the mula group consisting of zero and the positive integer 000R 1,to form a keto aldehyde having the formula CHO-CHOH-CHz-N oomnonomcmo0),.OR'

c 0 0 R A: aryl- 02 0110-0 OCHz-NOCO(N HCH2CH2COL|OR oxidizing thehydroxy aldehyde to form a keto W140. aldehyde having the formula and,subsequently, reacting the latter compound COOR with2,4,5-triamino-fi-hydroxypyrimidine to form 0110-00--OHz-NOO(NHLHCH;CH:C 0)..0R' a compound having a formula aryl s 02 Rc0012 and, subsequently, reacting the latter compound C with 2,4,5triamino 6 hydroxypyrimidine to N CH! IFOCWNHAHCHZCE (MOE form acompound having the formula H Nk aryl-SO:

22. The method of claim 21 wherein the acetal 5 aryls 02 is ethyl N-(3,3diethoxy 2 ketopropyl) -N-(ptoluenesulfonyl) -p-aminobenzoate.

23. The method of claim 21 wherein the acetal g fi mi g g zz g gfi gg gthe 1s methyl N '(33'dletpoxya'ketopropyl) droxy acetal is ethylN-(3,3-diethoxy-2-hydroxytoluenesulfonyl)-p-ammobenzoyl)-glutamate.propyl) N (p toluenesulfonyl) p ammm 24. The method which includes:oxidizing a benzoate. hydroxy aldehyde havmg the gg gz 31. The method ofclaim 29 wherein the hy- & droxy acetal is diethyl N (N (3,3 diethoxy-GHOCHOH'CHZN CWNH HCHQOHZCOMOR 2 hydroxypropyl) N (p toluenesulfonyl)-arm-so; p-aminobenzoyl)-glutamate.

OH N o o 0 R R and n having the values given. l; l T I O (NHJJHOEHCHKJ.0R'

H2N\N N/ 23'. ""32. The method which includes: oxidizing a keto alcoholhaving the formula aryl-SO: wherein R is from the group consisting ofhydrogen and the alkyl radicals and n is from the group consisting ofzero and the positive integer l, to form a keto aldehyde having theformula :7 aryl-SO: and, subsequently, reacting thelatter compound with2,4,5 triamino 6 hydroxypyrimidine to form a compound having the formulaicon \ om-N coma HCHzCEhC-OL-OR' J 3 aryl-S O2 R and n having the valuesgiven.

33. The method of claim 32 wherein the keto alcohol is oxidized withcupric acetate.

34. The method of claim 32 wherein the keto alcohol is oxidized withchromic acetate.

35. The method of claim- 32 wherein the keto alcohol is ethyl N (3hydroxy 2 ketopropyl) N (p-- toluenesulfonyl) p aminobenzoate.

36. The method of claim 32 wherein the keto alcohol is diethyl N (N (3hydroxy 2 ketopropyl) N (p 4 toluenesulfonyl) p aminobenzoyl)-glutamate.

3'7. The method which includes: oxidizing a methoxy ketone having theformula 0 00B omoon-o o-om-NOc omncnomcmo 0 ,.0 R

aryl- 02 wherein R. is from the group consisting of hydrogen and thealkyl radicals and n is from the 24; group consisting of zero and thepositive integer 1, to form a keto aldehyde having the formula v o 0 ORaryl- Oz and, subsequently, reacting the latter with 2,4,5-triamino-S-hydroxypyrimidine to form a compound'having the formulacnz-Nooomuencmomc0),.0R HNL l aryl-Oz REFERENCES CITED The followingreferences are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,211,467 Kimball et al. Aug. 13,1940 2,444,002 -Boothe June 22, 1948 2,444,005 Cosulich June 22, 19482,465,316 Maury Mar. 22, 1949 OTHER REFERENCES Lederle Bulletin, 13 (NO.3), 21 (1948).

29. THE METHOD WHICH INCLUDES: HYDROLYZING A HGYDROXY ACETAL HAVING THEFORMULA